1. Field of the Invention
This invention relates to a peptide suitable for eliciting an immune response against forms of Gonadotropine Releasing Hormone (GnRH) also referred to as Luteinizing Hormone Releasing Hormone (LHRH). The invention further relates to immunogenic compositions and vaccines, pharmaceuticals, and other medicinal preparations based on such a peptide. The invention further relates to the use of such a vaccine or medicinal preparation in a method of immunizing a mammal against GnRH to influence reproductive or behavioral characteristics of that mammal and in a method of improving the carcass quality of pigs. The invention also relates to a peptide suitable for eliciting a selective immunogenic response against GnRH-I or GnRH-II. Further the invention relates to antibodies against GnRH-I, to compositions comprising these antibodies and to the use of the peptides in pharmaceutical compositions or in the preparation for a medicament for the treatment of prostate cancer.
2. State of the Art
GnRH-I (in the literature generally depicted as GnRH) is a small 10 amino acid long peptide (decapeptide) from the hypothalamus. The amino acid sequence of GnRH-I (SEQ ID NO:1) can be represented by the following three-letter code:
pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2
or the corresponding one letter code where pE is pyroglutamic acid and # is amide:
pE H W S Y G L R P G#xe2x80x83xe2x80x83SEQ ID NO:1
GnRH-I acts at the hypophysis to cause an increase in release of biologically active Follicle-Stimulating Hormone (FSH) and Luteinizing Hormone (LH) in the blood, which in turn stimulate the development of the tests in the growing made animal and the synthesis of male steroids. In the growing female animal the development of the ovaries is stimulated, as is the development of follicles within the ovary, synthesis of female steroids, and ovulation.
It is known that GnRH-I, if coupled to a carrier protein, can be used to vaccinate animals. Such a vaccination can be carried out for various reasons, all of which are connected with the natural function of the GnRH-I. As is known, a drastic reduction of LH and/or FSH in the blood inhibits the production of male steroids or androgens and sperm in the testis of the male and the formation of female steroids or progestagens and estrogens and follicle maturation in the ovary of the female. Such a reduction in the amounts of androgens, progestagens and estrogens in the blood, to a level comparable to that obtainable by removing the testes or ovaries via castration, can be achieved by effective immunization of the animal against GnRH-I. In male animals, in many cases the testes then appear to develop slowly or not at all, with no synthesis of androgens (male steroid hormones) and no formation of spermatozoa. In female animals the activity of the ovaria appears to diminish, with no synthesis of estrogens and progestagens (female steroid hormones), and inhibition of ripening of follicles and ovulation.
Recently it was reported that a second form of GnRH (GnRH-II) is present in primate brain (Lescheid et al. Endocrinol. 138(1997) 5618-5629) and a gene for this second GnRH molecule was cloned from a human genomic library (GnRH-II, amino acid sequence pEHWSHGWYPG# (SEQ ID NO:2)) (White et al., PNAS USA 95 (1998) 305-309). Mammalian GnRH-I (SEQ ID NO 1) is hardly expressed outside the brain. A few exceptions are known in this respect. GnRH I is present in the endometrium of women with a menstrual cycle (Casan et al. Fertil, Sateril, 1998, 70, 102-106) and is expressed during pregnancy in the human placenta (Kelly et al. DNA cell Biol. 1991, 10, 411-421). GnRH and mRNA was found in ovary, testis, thymus, placenta and hypothalamus of the rat (Oikawa et al., Endocrinology, 1990, 127, 2350-2356). Expression of GnRH was detected in immune tissue (spleen, thymus and lymphocytes) of pigs (Weesner et al., Life Sci, 1997, 61, 1643-1649).
GnRH-II is expressed in many tissues outside the brain, and is found is especially high concentrations in the kidneys, bone marrow and prostate. The presence of GnRH-II in diverse tissues other than the brain suggests that GnRH-II may have multiple functions. In addition, the strictly conserved structure of the GnRH-II peptide throughout diverse vertebrate species suggests that this neuropeptide possesses vital bioactivites. Until now, however, the functions of GnRH-II have been practically unknown. Several types of differential lymphocytes, such as T- and B-lymphocytes and mast cells, produce GnRH and GnRH-like peptides. Significant numbers of the latter cell type are present in kidney, bone marrow and prostate, perhaps contributing to the high GnRH-II expression in these tissues. GnRH II seems less involved in reproduction as compared to GnRH-I. In the hypogonadal mouse, mouse which lack the GnRH-I gene, GnRH-II producing cells are present in the same distribution as in normal mouse, but this is not sufficient to cause normal gonadal development in these mouse (Chen et al. FEBS Letters 435 (1998) 199-203). However, macaques in luteal phase of the menstrual cycle showed a marked increase in plasma luteinizing hormone concentrations after intravenous administration of GnRH-II, but this increase could not be induced during the mid follicular phase (Lescheid et al., Endocrinol. 138 (1997) 5618-5629.)
The invention now provides the insight that by providing peptide sequences that allow discrimination between the different types of GnRH, more adequate and efficient use can be made of the variation or different in immunological response to the different types of GnRH. More particularly, the invention provides the insight that improvements in the efficiency and selectivity of the vaccines against GnRH-I can be achieved.
Immunization against GnRH-I is effective in neutralizing GnRH-I and results in reduced genadotropin levels and blocking of gonadal steroid synthesis. However, nothing in known about any physiological effects of the antibodies raised against GnRH-I on the function of GnRH-II. As GnRH-II is mainly synthesized and secreted in the kidneys antibodies raised against GnRH-I that cross-react with GnRH-II may affect kidney function. To obviate possible side effects of GmRH-I immunization on kidney function it would be desirable to direct the antigenic response of an immunocastration vaccine specifically toward GnRH-I and to avoid possible harmful side-effects due to neutralization of non-gonadal GnRH-II.
If the reproductive capacity alone, often with its accompanying sexual behavior, of a species needs to be annulled, it would be preferred to aim at an immunocastration vaccine specifically neutralizing GnRH-I. Hence the desired to come to a selective immunization against Gonadotropine Releasing Hormone(s), preferably selective against GnRH-I.
In veterinary medicine, 100% effective immunization against GnRH-I could be used for the sterilization of, e.g., small domestic animals such as male and female cats and dogs, or for the treatment of aggressiveness in male dogs and bulls, simply by vaccination instead of by drastic surgery such as castration or ovariectomy. Other conceivable reasons for immunization against GnRH-I are to prevent heat in female animals, such as dogs, cats and cows, and restlessness in male animals being fattened by slaughter.
In human health care immunization against GnRH-I and/or GnRH-II can be used in the treatment of prostatic cancer and breast cancer and, if required, in the treatment of some forms of pituitary carcinoma. In the case of prostate cancer it might be more desirable to neutralize both GnRH-I and GnRH-II, as the latter isoform is also highly expressed in prostate tissue.
Another use of a vaccine against GnRH-I is in the field of stock breeding, particularly the fattening of pigs of slaughter. The meat of male, sexually mature pigs (boars) has a typical odor, the so-called boar taint or boar odor. In the testes of the sexually mature pig, many C19-delta-16 steroids are formed which are stored in the fat tissue of the animal (Patterson, J. Sci. Food Agric. 19, 31-38 (1968); Brooks en Pearson, J. Anim. Sci. 62, 632-645 (1986); Claus, Zeitstchrift. Tierzxc3xcchtg. Zxc3xcchtungsbiol. 93, 38-47 (1976); Claus, Acta Endocrinol. (Copenh.) 91, Suppl. 225, 432-433 (1979)). These steroids are mainly responsible for the formation of the disagreeable urine-like odor when the meat is heated (Fuchs, Swedish J. Agric. Res. 1, 233-237 (1971); Bonneau, Livest, Prod. Sci. 9, 687-705 (1982)). Owing to this unpleasant odor, meat of male sexually mature pigs is generally unsuitable for consumption and unfit for export. Because about 10% of the male slaughter pigs are already sexually mature before the slaughter time, this potentially entails a great loss for the pig farming industry.
In order to control and prevent these losses, nearly all male piglets are castrated when they are young, with a surgical procedure that is generally executed without any form of anaesthesia. Apart from the animal unfriendly aspect of such a castration, castration also leads to infections, growth inhibition, and a final carcass quality inferior to that of an intact animal, at least as long as that intact animal has not yet developed boar taint (Walstra, Livest. Prod. Sci. 1, 187-96 (1974)).
An animal friendly alternative consists in the reduction of the GnRH-I concentration in the pig pituitary by means of immunization against GnRH-I, the so-called immunocastration. This reduction in GnRH-I levels leads to a reduction in the concentrations of biologically active FSH and LH, which in turn will inhibit development of the testes in the growing animals and inhibit the synthesis of testicular steroids, including androstenone, testosterone and estrogens. This method prevents the occurrence of boar taint in male pigs at slaughter time and makes surgical castration unnecessary as androstenone levels are reduced to levels low or undetectable (Oonk et al., 1995, Livestock production Science 42, 63-71).
A strict requirement for an acceptable vaccine against boar taint is that in almost all pigs development of the testes is delayed to such an extent that boar taint will not have occurred at the time of slaughter, and that in the case that the vaccine does not reduce testis development in an animal, this can be easily detected in a too large testis size in comparison to successfully immunocastrated pigs.
In the existing literature and previous patent applications regarding the anti-fertility properties of vaccines against GnRH-I, the results of vaccinations often appear to be variable. In most of the described studies, either a small percentage of the vaccinated animals do not respond to the vaccination, or large doses of vaccine, multiple vaccinations or commercially unacceptable adjuvants are needed to produce the desired effect (Hoskinson et al., 1990, Austr. J. Biotech. 4, 166-170; Falvo et al., (1986) J. Anim. Sci. 63:986-994; Clarke et al., 1998, Endocrinology 139, 2007-2014; Adams T. E. and B. M. Adams, Feedlot performance of steers and bulls active immunized against Gonadotropine-Releasing Hormone, J. Anim. Sci. 1992, 70: 1691-1698; Brown et al., Immunization of sheep against GnRH-I early in life: effects of reproductive function and hormones in rams, Journal of reproduction and Fertility (1994) 101, 15-21; Ferro et al., Immunological castration using a Gonadotropine-releasing Hormone analogue conjugated to PPD, Food and agricultural immunology, 1995, 7, 259-272; U.S. Pat. No. 4,608,251; Int. patent appl. WO 88/05308).
Some studies suggested an efficacy of 100% of a vaccine against GnRH-I, but the vaccine was not tested in a large number of animals (Ladd et al. (1994), Development of an antifertility vaccine for pets based on active immunization against Luteinizing Hormone releasing hormone, Biology of Reproduction 51, 1076-1083; J. G. Manns and S. R. Robbins (1997). Prevention of boar taint with a recombinant based GnRH vaccine, In: Boar taint in entire male pigs, Proceedings of a meeting of the EAAP working group xe2x80x9cProduction and Utilisation of Meat from Entire Male Pigsxe2x80x9d, EAAP Publication No. 92, 137-140;); other studies report the efficacy of the vaccine as the mean value of the treated animals, since individual values did not show a clear difference between immunized and untreated controls (Bonneau et al., J. Anim. Sci. 72, 14-20 (1994); Hennesy et al., 1997. Elimination of boar taint; a commercial boar taint vaccine for male pigs. In: Bonneau, M., Lundstrxc3x6m, K. and Malmfors, B. (Eds.), Boar taint in entire male pigs. Wageningen Pers, Wageningen, EAAP Publication No. 92, 141-145).
The difficulty in preparing this type of vaccines probably is caused by the phenomenon of tolerance. Self substances such as hormones are not recognised, as foreign but rather are tolerated by the immune system. Normally no antibodies are elicited against self substances. In order to a vaccine to be successful, it must be sufficiently foreign. Only when the vaccine is foreign enough will the immune system not tolerate the vaccine and the production of antibodies be induced. Conversely, however, the antibodies must still be capable of recognizing the hormone, and thus the vaccine cannot be too xe2x80x98foreignxe2x80x99.
As these conditions appear to be mutually exclusive, it was not certain, until recently, if such substances could be prepared at all. One attempt to produce GnRH-like peptide vaccines consisted of the replacement of Gly at position 6 of the GnRH-I decapeptide by a dextrorotary amino acid (D-Tyrp; Chaffaux et al., Recueil de Mxc3xa9dicine Vxc3xa9txc3xa9rinaire 161 (2), 133-145, 1985). It was, however, demonstrated that a vaccine preparation containing this modified GnRH-peptide performed even less well than the normal GnRH-I decapeptide (European Patent application 464, 124 A).
Recently, we have shown definitively that it is possible to elicit an effective antibody response in all individuals vaccinated against GnRH-I (Meloen et al., Vaccine 12, 741-746 (1994)). In these experiments pigs were vaccinated twice with an GnRH-I vaccine that departs from the classical type of GnRH-I vaccine (GnRH-I coupled to a carrier protein, in Freund""s adjuvant) namely the tandem-GnRH-I vaccine (European patent nr. 0464124). In this publication a peptide is described which is characterized in that is comprises at least 2 GnRH-I sequences in tandem (SEQ ID NO:3) according to the general formula
Z1-Glx-His-Trp1-Ser-Tyr-Gly-Leu-Arg-Pro[-Gly-X-Gln-His-Trp2-Ser-Tyr-Gly-Leu-Arg-Pro]n-Gly-Z2,
in which amino acids are designated according to the three-letter code, Trp1 and Trp2 are tryptophan (trp) or formylated tryptophan (N(indole)-formyl-tryptophan), n is a number having a value of at least 1, X is either a direct bond or a spacer group between the amino acids Gly and Gln, Z1-Glx is either pGlu (pyroglutamic acid) or Gln having attached thereto a tail comprising one or more additional amino acids, and Gly-Z2 is either Gly-NH2 or Gly having attached thereto a tail comprising one or more additional amino acids. In this general formula, X may be a direct bond between the amino acids glycine and glutamine, i.e. these amino acids are interconnected directly without an intermediate link (via the normal peptide bond). The tandem-GnRH-I vaccine invention also comprises peptides in which the GnRH-I sequences are interconnected via spacers. The nature of the spacer group may vary greatly, from one or more amino acids to a shorter or longer hydrocarbon chain and other compound groups or molecules. In the above general formula, Z1-Glx preferably stands for pGlu (pyroglutamic acid), but can also stand for Gln having attached thereto a tail comprising one or more additional amino acids, e.g., to be used for coupling of the peptide to a carrier protein. In the above general formula, Gly-Z2 stands for, e.g., Gly-NH2, or Gly having attached thereto a tail comprising one or more additional amino acids, e.g., to be used for coupling of the peptide to a carrier protein. Preferably, Gly-Z2 stands for Gly-Cys-NH2, the C terminal cysteine being added in connection with a possible coupling of the peptide to a carrier protein.
From WO 96/40755 it is known that the tandem-dimer principle applied to a variant of the GnRH-I molecule resulted in a vaccine that was highly effective in several mild adjuvants, namely Specol and a double oil emulsion, and was also effective in low doses. In this case, the variant of the GnRH-I molecule was formed by substitution of the sixth amino acid Gly of the decapeptide by a dextrorotatory (D-) amino acid, D-Lys, following which the resulting peptide was dimerised and coupled to a common carrier compound, ovalbumine. Thus, whereas a vaccine using D-amino acid substitutions of Gly at positions 6 of the original and a single GnRH-I decapeptide with a D-amino acid decreased the immunogenicity as compared to the original GnRH-I sequence (Chaffaux et al., Recueil de Mxc3xa9dicine Vxc3xa9txc3xa9rinaire 161 (2), 133-145, 1985), such substitutions with a D-amino acid applied to a tandem-dimer GnRH-I vaccine were able to generate even ore immunogenic GnRH-I vaccine preparations, Nevertheless, the method for vaccination required a repeat dosage of the vaccine in order to be completely effective. The necessity of an additional booster dosage in order to achieve essentially 100% vaccination of mammals against GnRH-I is a disadvantage of the known peptides. We also found that in certain cases use of the (D-Lys6)GnRH I tandem dimer (i.e. the GnRH I tandem dimer, with and without the D-Lys6 replacement) resulted in very low but still measurable amounts of testosterone, which is undesirable and a disadvantage of the (D-Lys6) GnRH I tandem dimer.
The present invention provides peptide sequences and that provide alternatives to the tandem D-Lys6 GnRH-I when applied in vaccines that result in vaccines that are effective for immunocastration.
An aspect of the present invention is the determination of the extent to which the amino acids in the tandem GnRH-I sequence can be varied while the resulting substituted tandem GnRH-I is still able to produce an immunogenic response to GnRH-I sufficient for immunocastration. Thus, the invention provides for the generation of a peptide sequence that can induce the production of antibodies against GnRH-I, which are also sufficiently competitive, both in amount and activity.
A further aspect of the present invention is to provide to a peptide sequence that selectively induces the production of antibodies against GnRH-I, while inducing little or no immune response towards GnRH-II. A preferred embodiment thereof is a peptide sequence that not only selectively induces the production of antibodies against GnRH-I but is also effective in immunocastration, while an immune response to GnRH-II is reduced or absent.
The present inventors have found that in the tandem GnRH-I peptide sequence various amino acids can be replaced, resulting in a decreasing resemblance to the self-hormone while at the same time retaining or even increasing the ability of the peptide to elicit GnRH-I binding antibodies. Also, certain replacements of amino acids in the GnRH-I peptide sequence result in a selective immune response towards GnRH-I and to a reduced or absent immunoresponse towards GnRH-II.
In an aspect of the invention, certain modified tandem GnRH-I peptide sequences provide for vaccines that are not only capable of reducing testes growth in male animals but are also capable of essentially reducing testosterone levels to a degree that they can not be determined by conventional techniques.
Further, vaccines prepared from the these peptide-sequences express an activity that in most cases eliminated the need for the second booster immunization, as with the conventional D-Lys6 tandem GnRH-I, to achieve essentially 100% activity. An activity or efficacy of 100% in the terms of the present invention is defined as a testosterone level that is essentially undetectable with conventional techniques after a single vaccination.
One of the most notable features of the present invention is that antibodies raised by these alternative GnRH vaccines discriminate between GnRH-I and GnRH-II. Thus peptides according to the invention express an increased or retained activity against GnRH-I, while at the same time a reduced or absent immune response to GnRH-II is found. This allows for the development of peptides that express an inverse effect, that is, they express an increased or retained activity against GnRH-II while at the same time expressing a reduced or absent immune response against GnRH-I.
The invention relates in one aspect to a peptide that comprises a modified tandem GnRH-I decapeptide sequence whereby vaccination with the peptide in a suitable dosage allows for a testosterone level that is essentially non-detectable.
The invention also relates to a peptide that comprises at least two coupled GnRH-I decapeptide sequences, optionally coupled through a spacer, which allows for an immunogenic response that allows for the effective discrimination between GnRH-I and GnRH-II.
The peptides according to the invention are sufficiently like the hormone but at the same more xe2x80x98foreignxe2x80x99 to the immune system and have an increased capability to induce the production of antibodies directed against the hormone.
A feature of the invention is that individual tandem units can be dimerised to further enhance its immunogenicity without losing the possibility of coupling the peptide or peptide composition to a carrier compound protein.
The techniques for dimerisation and coupling of the tandem to a carrier similar to those described in WO 96/40755 may be used. It is also envisioned that peptides containing only a portion of the GnRH-I or II peptide sequences can be used in the present invention. Examples thereof are nonapeptides and undecapeptides.
The linkers for use in the peptides according to the invention can be selected from the linkers described elsewhere in this application or linkers and such as 
Linkers are used for coupling two or more dimerised peptide sequences. The amino acid which is used to replace the amino acid in the tandem peptide sequences is preferably an amino acid which is a relative simple one, such as alanine. In a preferred embodiment therefor the different amino acid is alanine. Other amino acids can also be used to replace the amino acid in the tandem decapeptide sequence. Preferably only conservative replacements are carried out. Conservative replacements are amino acid substitutions in which bulky amino acids are replaced by bulky amino acids, aromatic amino acids by aromatic amino acids etc. These concepts are well known to those skilled in the art.
Spacers can be placed between the peptides according to the invention. This allows for the formation of multimers. Suitable spacers are known in the art.